Antibody (11C7) anti Nogo-A and its pharmaceutical use

ABSTRACT

This invention relates to molecules, such as for example monoclonal antibodies or Fab fragments thereof, which are capable of binding to the human NogoA polypeptide or human NiG or human NiG- or human NogoA_623-640 with a dissociation constant, 1000 nM; polynucleotides encoding such a binding molecule; an expression vector comprising such polynucleotides; the use of such a binding molecule in the treatment of nerve repair, a pharmaceutical composition comprising such a binding molecule; and to a method of treatment of diseases associated with nerve repair.

RELATED APPLICATIONS

This present invention is a divisional patent application of U.S. patentapplication Ser. No. 10/538,201, now U.S. Pat. No. 7,785,593, filed onMar. 8, 2008, which claims priority to PCT application numberPCT/EP2003/013960, filed Dec. 9, 2003, which claims priority to GreatBritain application No. 0228832.2, filed on Dec. 10, 2002, the entiretyof which applications are herein incorporated by reference.

This invention relates to NogoA binding molecules, such as for examplemonoclonal antibodies or Fab fragments thereof.

Neuronal regeneration following injury in the adult central nervoussystem (CNS) is limited due to the presence of the inhibitory myelinenvironment that ensheaths axons and formation of scar tissue. In thelast few years important insights have been gained into the molecularunderstanding why the CNS is unable to spontaneously repair itselffollowing injury. Inhibitory molecules in the myelin are the majorimpediment for the axonal regeneration, particularly immediately afterthe injury. So far NogoA, Myelin-Associated Glycoprotein (MAG) andmyelin-oligodendrocyte glycoprotein (OMgp) have been characterised aspotent inhibitors of neurite outgrowth. In addition, myelin alsocontains other inhibitory components, such as, chondroitin sulphateproteoglycans. Nogo-A is a member of the reticulon protein family and ithas at least two biologically active and pharmacologically distinctdomains termed Amino-Nogo and Nogo-66. While the receptor site for theformer is not known so far, Nogo-66 inhibits neuronal growth in vitroand in vivo via the neuronal receptor NgR. In addition to Nogo-66, MAGand OMgp also bind to the NgR with high affinity and inhibit neuriteoutgrowth.

Potential new research approaches currently pursued for enhancement ofnerve repair include digestion of scar tissue using an enzymechondroitinase ABC, bridging techniques using Olfactory ensheathingcells and stem cells and protein growth factors to boost neuronalgrowth. Blocking actions of neurite outgrowth inhibitors by modulationof intracellular signalling mediators such as Rho, a membrane-boundguanosine trisphosphatase (GTPase), which appears to be a key link inthe inhibition of axonal growth. Cyclic adenosine monophosphate (cAMP)which can overcome myelin associated inhibition in vitro and induceregeneration in vivo. Use of peptide inhibitor of the NgR receptor (NEP1-40) to induce neuronal regrowth and functional recovery in ratsfollowing spinal injury.

In addition to the use of the approaches described above, attention hasalso focused upon the use of certain monoclonal antibodies to neutralizeneurite growth inhibitory molecules of the central and peripheralnervous system, in particular to neutralize the neurite growthinhibitory activity of NogoA. Thus it has been shown that the monoclonalantibody IN-1 or the IN-1 Fab fragment thereof induce neurite outgrowthin vitro and enhance sprouting and regeneration in vivo (Schwab M E etal. (1996) Physiol. Rev. 76, 319-370). Testing different domains of theNogoA for neurite growth inhibitory activity have delineated severalinhibitory domains in the molecule (Chen et al. (2000) Nature 403,434-439; GrandPre et al. (2000) Nature 403, 439-444; Prinjha et al.(2000) Nature 403, 383-384; see also detailed analysis in Example 1).

Natural immunoglobulins or antibodies comprise a generally Y-shapedmultimeric molecule having an antigen-binding site at the end of eachupper arm. The remainder of the structure, in particular the stem of theY mediates effector functions associated with the immunoglobulins.Antibodies consists of a 2 heavy and 2 light chains. Both heavy andlight chains comprise a variable domain and a constant part. An antigenbinding site consists of the variable domain of a heavy chain associatedwith the variable domain of a light chain. The variable domains of theheavy and light chains have the same general structure. Moreparticularly, the antigen binding characteristics of an antibody areessentially determined by 3 specific; regions in the variable domain ofthe heavy and light chains which are called hypervariable regions orcomplementarity determining regions (CDRs). These 3 hypervariableregions alternate with 4 framework regions (FRs) whose sequences arerelatively conserved and which are not directly involved in binding. TheCDRs form loops and are held in close proximity by the framework regionswhich largely adopt a 6-sheet conformation. The CDRs of a heavy chaintogether with the CDRs of the associated light chain essentiallyconstitute the antigen binding site of the antibody molecule. Thedetermination as to what constitutes an FR or a CDR region is usuallymade by comparing the amino acid sequence of a number of antibodiesraised in the same species. The general rules for identifying the CDRand FR regions are general knowledge of a man skilled in the art and canfor example be found in the “Abysis” Antibodies Database operated byUniversity College London.

It has now surprisingly been found that a novel monoclonal mouseantibody (hereinafter called “11C7”) raised against a polypeptidefragment of rat NogoA (SEQ ID NO: 1) and of the IgG1 type has betterproperties than the NogoA antibodies of the prior art especially withregard to the binding affinity to NogoA of different species includingthe homo sapiens and with regard to its higher NogoA neurite outgrowthneutralizing activity at a given antibody concentration. Moreover it isnow possible to construct other NogoA binding molecules having the samehypervariable regions as the said antibody.

Accordingly, the invention provides binding molecules to a particularregion or epitope of NogoA (hereinafter referred to as “the BindingMolecules of the invention” or simply “Binding Molecules”). Preferablythe Binding Molecules of the invention bind to human NogoA_(—)623-640(orthologous fragment against which 11C7 was raised; =SEQ ID NO: 6),human Nig-D20 (orthologous to the smallest fragment of NogoA withneurite outgrowth inhibitory activity, SEQ ID NO: 24), human NogoA (SEQID NO: 5) or human NiG (which is the most potent neurite outgrowthinhibitory fragment of NogoA and starts at amino acid No. 186 and endsat amino acid No. 1004 of human NogoA, =SEQ ID NO: 5) with adissociation constant (Kd)<1000 nM, more preferably with a Kd<100 nM,most preferably with a Kd<10 nM. The binding reaction may be shown bystandard methods (qualitative assays) including, for example, the ELISAmethod described in Example 6 and the biosensor affinity methoddescribed in the example 7. In addition, the binding to human NogoA andalmost more importantly the efficiency may be shown in a neuriteoutgrowth assay, e.g. as described below.

Thus, in a further preferred embodiment the Binding Molecules (at aconcentration of 1 mg/ml, more preferably at 0.1 mg/ml even morepreferably at 0.01 mg/ml culture medium) enhance the number of neuritesof rat cerebellar granule cells on a substrate of rat spinal cordprotein extract by at least 20%, preferably 50%, most preferred 100%compared to the number of neurites of rat cerebellar granule cells whichare treated with a control antibody that does not bind to the humanNogoA, human NiG, human Nig-D20 or NogoA_(—)623-640 polypeptide (i.e.that has a dissociation constant >1000 nM).

In a further preferred embodiment the Binding Molecules of the inventioncomprises at least one antigen binding site, said antigen binding sitecomprising in sequence, the hypervariable regions CDR1-11C7, CDR2-11C7and CDR3-11C7; said CDR1-11C7 having the amino acid sequence SEQ ID NO:8, said CDR2-11C7 having the amino acid sequence SEQ ID NO: 9, and saidCDR3-11C7 having the amino acid sequence SEQ ID NO: 10; and directequivalents thereof.

In a further aspect of the invention, the Binding Molecule of theinvention comprises at least one antigen binding site, said antigenbinding site comprising either

-   a) in sequence the hypervariable regions CDR1-11C7, CDR2-11C7 and    CDR3-11C7; said CDR1-11C7 having the amino acid sequence of SEQ ID    NO: 8, said CDR2-11C7 having the amino acid sequence of SEQ ID NO:    9, and said CDR3-11C7 having the amino acid sequence SEQ ID NO: 10;    or-   b) in sequence the hypervariable regions CDR1′-11C7, CDR2′-11C7 and    CDR3′-11C7, said CDR1′-11C7 having the amino acid sequence of SEQ ID    NO: 11, said CDR2′-11C7 having the amino acid sequence of SEQ ID NO:    12, and said CDR3′-11C7 having the amino acid sequence of SEQ ID NO:    13; or-   c) direct equivalents thereof.

In a further aspect of the invention, the Binding Molecule of theinvention comprises at least

-   a) a first domain comprising in sequence the hypervariable regions    CDR1-11C7, CDR2-11C7 and CDR3-11C7; said CDR1-11C7 having the amino    acid sequence of SEQ ID NO: 8, said CDR2-11C7 having the amino acid    sequence of SEQ ID NO: 9, and said CDR3-11C7 having the amino acid    sequence SEQ ID NO: 10; and-   b) a second domain comprising in sequence the hypervariable regions    CDR1′-11C7, CDR2′-11C7 and CDR3′-11C7, said CDR1′-11C7 having the    amino acid sequence of SEQ ID NO: 11, said CDR2′-11C7 having the    amino acid sequence of SEQ ID NO: 12, and said CDR3′-11C7 having the    amino acid sequence of SEQ ID NO: 13; or-   c) direct equivalents thereof.

Moreover, the invention also provides the following Binding Molecule ofthe invention, which comprises at least one antigen binding sitecomprising

-   a) either the variable part of the heavy chain of 11C7 (SEQ ID NO:    2); or-   b) the variable part of the light chain of 11C7 (SEQ ID NO: 3), or    direct equivalents thereof.

When the antigen binding site comprises both the first and seconddomains, these may be located on the same polypeptide molecule or,preferably, each domain may be on a different chain, the first domainbeing part of an immunoglobulin heavy chain or fragment thereof and thesecond domain being part of an immunoglobulin light chain or fragmentthereof.

Examples of Binding Molecules of the invention include antibodies asproduced by B-cells or hybridomas and chimeric or humanized antibodiesor any fragment thereof, e.g. F(ab′)₂; and Fab fragments, as well assingle chain or single domain antibodies.

A single chain antibody consists of the variable domains of an antibodyheavy and light chains covalently bound by a peptide linker usuallyconsisting of from 10 to 30 amino acids, preferably from 15 to 25 aminoacids. Therefore, such a structure does not include the constant part ofthe heavy and light chains and it is believed that the small peptidespacer should be less antigenic than a whole constant part. By “chimericantibody” is meant an antibody in which the constant regions of heavy orlight chains or both are of human origin while the variable domains ofboth heavy and light chains are of non-human (e.g. murine) origin. By“humanized antibody” is meant an antibody in which the hypervariableregions (CDRs) are of non-human (e.g. murine) origin, while all orsubstantially all the other parts of the immunoglobulin e.g. theconstant regions and the highly conserved parts of the variable domains,i.e. the framework regions, are of human origin. A humanized antibodymay however retain a few amino acids of the murine sequence in the partsof the framework regions adjacent to the hypervariable regions.

Hypervariable regions may be associated with any kind of frameworkregions, preferably of murine or human origin. Suitable frameworkregions are described in “Sequences of proteins of immunologicalinterest”, Kabat E. A. et al, US department of health and humanservices, Public health service, National Institute of Health.Preferably the constant part of a human heavy chain of the BindingMolecules may be of the IgG4 type, including subtypes, preferably theconstant part of a human light chain may be of the κ or λ type, morepreferably of the κ type.

Monoclonal antibodies raised against a protein naturally found in allhumans may be developed in a non-human system e.g. in mice. As a directconsequence of this, a xenogenic antibody as produced by a hybridoma,when administered to humans, elicits an undesirable immune response,which is predominantly mediated by the constant part of the xenogenicimmunoglobulin. This clearly limits the use of such antibodies as theycannot be administered over a prolonged period of time. Therefore it isparticularly preferred to use single chain, single domain, chimeric orhumanized antibodies which are not likely to elicit a substantialallogenic response when administered to humans.

In view of the foregoing, a more preferred Binding Molecule of theinvention is selected from a chimeric antibody, which comprises at least

-   a) one immunoglobulin heavy chain or fragment thereof which    comprises (i) a variable domain comprising in sequence the    hypervariable regions CDR1-11C7, CDR2-11C7 and CDR3-11C7 and (ii)    the constant part or fragment thereof of a human heavy chain; said    CDR1-11C7 having the amino acid sequence (SEQ ID NO: 8), said    CDR2-11C7 having the amino acid sequence (SEQ ID NO: 9), and said    CDR3-11C7 having the amino acid sequence (SEQ ID NO: 10), and-   b) one immunoglobulin light chain or fragment thereof which    comprises (i) a variable domain comprising in sequence the    hypervariable regions CDR1′-11C7, CDR2′-11C7 and CDR3′-11C7 and (ii)    the constant part or fragment thereof of a human light chain; said    CDR1′-11C7 having the amino acid sequence (SEQ ID NO: 11), said    CDR2′-11C7 having the amino acid sequence (SEQ ID NO: 12), and said    CDR3′-11C7 having the amino acid sequence (SEQ ID NO: 13); or    direct equivalents thereof.

Alternatively, a Binding Molecule of the invention may be selected froma single chain binding molecule which comprises an antigen binding sitecomprising

-   a) a first domain comprising in sequence the hypervariable    CDR1-11C7, CDR2-11C7 and CDR3-11C7; said CDR1-11C7 having the amino    acid sequence (SEQ ID NO: 8), said CDR2-11C7 having the amino acid    sequence (SEQ ID NO: 9), and said CDR3-11C7 having the amino acid    sequence (SEQ ID NO: 10); and-   b) a second domain comprising in sequence the hypervariable    CDR1′-11C7, CDR2′-11C7 and CDR3′-11C7; said CDR1′-11C7 having the    amino acid sequence (SEQ ID NO: 11), said CDR2′-11C7 having the    amino acid sequence (SEQ ID NO: 12), and said CDR3′-11C7 having the    amino acid sequence (SEQ ID NO: 13); and-   c) a peptide linker which is bound either to the N-terminal    extremity of the first domain and to the C-terminal extremity of the    second domain or to the C-terminal extremity of the first domain and    to the N-terminal extremity of second domain;    or direct equivalents thereof.

As it is well known, minor changes in an amino acid sequence such asdeletion, addition or substitution of one or several amino acids maylead to an allelic form of the original protein which has substantiallyidentical properties. Thus, by the term “direct equivalents thereof” ismeant either any single domain Binding Molecule of the invention(molecule X)

-   (i) in which each of the hypervariable regions CDR1, CDR2, and CDR3    of the Binding Molecule is at least 50 or 80% homologous, preferably    at least 90% homologous, more preferably at least 95, 96, 97, 98,    99% homologous to the equivalent hypervariable regions of CDR1-11C7    (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO:    10), whereas CDR1 is equivalent to CDR1-11C7, CDR2 is equivalent to    CDR2-11C7, CDR3 is equivalent to CDR3-11C7; and-   (ii) which is capable of binding to the human NogoA, human NiG,    human NiG-D20, or human NogoA_(—)623-640, preferably with a    dissociation constant (Kd)<1000 nM, more preferably with a Kd<100    nM, most preferably with a Kd<10 nM, or    any binding molecule of the invention having at least two domains    per binding site (molecule X′)-   (iii) in which each of the hypervariable regions CDR1, CDR2, CDR3,    CDR1′, CDR2′ and CDR3′ is at least 50 or 80% homologous, preferably    at least 90% homologous, more preferably at least 95, 96, 97, 98,    99% identical to the equivalent hypervariable regions of CDR1-11C7    (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9), CDR3-11C7 (SEQ ID NO: 10),    CDR1′-11C7 (SEQ ID NO: 11), CDR2′-11C7 (SEQ ID NO: 12), and    CDR3′-11C7 (SEQ ID NO: 13), whereas CDR1 is equivalent to CDR1-11C7,    CDR2 is equivalent to CDR2-11C7, CDR3 is equivalent to CDR3-11C7,    CDR1′ is equivalent to CDR1′-11C7, CDR2′ is equivalent to    CDR2′-11C7, CDR3′ is equivalent to CDR3′-11C7; and-   (iv) which is capable of binding the human NogoA, human NiG, human    NiG-D20, or human NogoA_(—)623-640, preferably with a dissociation    constant (Kd)<1000 nM, more preferably with a Kd<100 nM, most    preferably with a Kd<10 nM.

Thus further embodiments of the inventions are for example a BindingMolecule which is capable of binding to the human NogoA, human NiG,human NiG-D20, or human NogoA_(—)623-640 with a dissociation constant<1000 nM and comprises at least one antigen binding site, said antigenbinding site comprising either

-   -   in sequence the hypervariable regions CDR1, CDR2, and CDR3, of        which each of the hypervariable regions are at least 50%,        preferably 80, 90, 95, 96, 97, 98, 99% homologous to their        equivalent hypervariable regions CDR1-11C7 (SEQ ID NO: 8),        CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); or    -   in sequence the hypervariable regions CDR1′, CDR2′, and CDR3′,        of whish each of the hypervariable regions are at least 50%,        preferably 80, 90, 95, 96, 97, 98, 99% homologous to their        equivalent hypervariable regions CDR1′-11C7 (SEQ ID NO: 11),        CDR2′-11C7 (SEQ ID NO: 12) and CDR3′-11C7 (SEQ ID NO: 13).

Furthermore, a Binding Molecule which is capable of binding the humanNogoA, human NiG, human NiG-D20, or human NogoA_(—)623-640 with adissociation constant <1000 nM and comprises

-   -   a first antigen binding site comprising in sequence the        hypervariable regions CDR1, CDR2, and CDR3, of which each of the        hypervariable regions are at least 50%, preferably 80, 90, 95,        96, 97, 98, 99% homologous to their equivalent hypervariable        regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and        CDR3-11C7 (SEQ ID NO: 10); and    -   a second antigen binding site comprising in sequence the        hypervariable regions CDR1′, CDR2′, and CDR3′, of which each of        the hypervariable regions are at least 50%, preferably 80, 90,        95, 96, 97, 98, 99% homologous to their equivalent hypervariable        regions CDR1′-11C7 (SEQ ID NO: 11), CDR2′-11C7 (SEQ ID NO: 12)        and CDR3′-11C7 (SEQ ID NO: 13).

This dissociation constant may be conveniently tested in various assaysincluding, for example, the biosensor affinity method described in theexample 7. In addition, the binding and functional effect of the BindingMolecules may be shown in a bioassay, e.g. as described below.

The constant part of a human heavy chain may be of the γ1; γ2; γ3; γ4;α1; α2; δ or ε type, preferably of the γ type, more preferably of theγ4; type, whereas the constant part of a human light chain may be of theκ or λ type (which includes the λ1; λ2; and λ3 subtypes) but ispreferably of the κ type. The amino acid sequence of all these constantparts are given in Kabat et al (Supra).

Conjugates of the binding molecules of the invention, e.g. enzyme ortoxin or radioisotope conjugates, are also included within the scope ofthe invention.

“Polypeptide”, if not otherwise specified herein, includes any peptideor protein comprising amino acids joined to each other by peptide bonds,having an amino acid sequence starting at the N-terminal extremity andending at the C-terminal extremity. Preferably the polypeptide of thepresent invention is a monoclonal antibody, more preferred is a chimeric(also called V-grafted) or humanized (also called CDR-grafted)monoclonal antibody. The humanized (CDR-grafted) monoclonal antibody mayor may not include further mutations introduced into the framework (FR)sequences of the acceptor antibody.

A functional derivative of a polypeptide as used herein includes amolecule having a qualitative biological activity in common with apolypeptide to the present invention, i.e. having the ability to bind tothe human NogoA, human NiG, human NiG-D20, or human NogoA_(—)623-640. Afunctional derivative includes fragments and peptide analogs of apolypeptide according to the present invention. Fragments compriseregions within the sequence of a polypeptide according to the presentinvention, e.g. of a specified sequence. The term “derivative” is usedto define amino acid sequence variants, and covalent modifications of apolypeptide according to the present invention, e.g. of a specifiedsequence. The functional derivatives of a polypeptide according to thepresent invention, e.g. of a specified sequence, e.g. of thehypervariable region of the light and the heavy chain, preferably haveat least about 65%, more preferably at least about 75%, even morepreferably at least about 85%, most preferably at least about 95, 96,97, 98, 99% overall sequence homology with the amino acid sequence of apolypeptide according to the present invention, e.g. of a specifiedsequence, and substantially retain the ability to bind the human NogoA,human NiG, human NiG-D20, or human NogoA_(—)623-640.

The term “covalent modification” includes modifications of a polypeptideaccording to the present invention, e.g. of a specified sequence; or afragment thereof with an organic proteinaceous or non-proteinaceousderivatizing agent, fusions to heterologous polypeptide sequences, andpost-translational modifications. Covalent modified polypeptides, e.g.of a specified sequence, still have the ability bind to the human NogoA,human NiG, human NiG-D20, or human NogoA_(—)623-640 by crosslinking.Covalent modifications are traditionally introduced by reacting targetedamino acid residues with an organic derivatizing agent that is capableof reacting with selected sides or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. Certain post-translational modifications are theresult of the action of recombinant host cells on the expressedpolypeptide. Glutaminyl and asparaginyl residues are frequentlypost-translationally deaminated to the corresponding glutamyl andaspartyl residues. Alternatively, these residues are deaminated undermildly acidic conditions. Other post-translational modifications includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl, tyrosine or threonyl residues, methylation of the α-aminogroups of lysine, arginine, and histidine side chains, see e.g. T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983). Covalent modifications e.g.include fusion proteins comprising a polypeptide according to thepresent invention, e.g. of a specified sequence and their amino acidsequence variants, such as immunoadhesins, and N-terminal fusions toheterologous signal sequences.

“Homology” with respect to a native polypeptide and its functionalderivative is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues of acorresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Neither N- or C-terminal extensions nor insertionsshall be construed as reducing identity or homology. Methods andcomputer programs for the alignment are well known.

“Amino acid(s)” refer to all naturally occurring L-α-amino acids, e.g.and including D-amino acids. The amino acids are identified by eitherthe well known single-letter or three-letter designations.

The term “amino acid sequence variant” refers to molecules with somedifferences in their amino acid sequences as compared to a polypeptideaccording to the present invention, e.g. of a specified sequence. Aminoacid sequence variants of a polypeptide according to the presentinvention, e.g. of a specified sequence, still have the ability to bindto human NogoA or human NiG or more preferably to NogoA_(—)623-640.Substitutional variants are those that have at least one amino acidresidue removed and a different amino acid inserted in its place at thesame position in a polypeptide according to the present invention, e.g.of a specified sequence. These substitutions may be single, where onlyone amino acid in the molecule has been substituted, or they may bemultiple, where two or more amino acids have been substituted in thesame molecule. Insertional variants are those with one or more aminoacids inserted immediately adjacent to an amino acid at a particularposition in a polypeptide according to the present invention, e.g. of aspecified sequence. Immediately adjacent to an amino acid meansconnected to either the ac carboxy or α-amino functional group of theamino acid. Deletional variants are those with one or more amino acidsin a polypeptide according to the present invention, e.g. of a specifiedsequence, removed. Ordinarily, deletional variants will have one or twoamino acids deleted in a particular region of the molecule.

A binding molecule of the invention may be produced by recombinant DNAtechniques in view of this, one or more DNA molecules encoding thebinding molecule must be constructed, placed under appropriate controlsequences and transferred into a suitable host organism for expression.

In a very general manner, there are accordingly provided

-   (i) DNA molecules encoding a single domain Binding Molecule of the    invention, a single chain Binding Molecule of the invention, a heavy    or light chain or fragments thereof of a Binding Molecule of the    invention; and-   (ii) the use of the DNA molecules of the invention for the    production of a Binding Molecule of the invention by recombinant    means.

The present state of the art is such that the skilled man will be ableto synthesize the DNA molecules of the invention given the informationprovided herein i.e. the amino acid sequences of the hypervariableregions and the DNA sequences coding for them. A method for constructinga variable domain gene is for example described in EP 239 400 and may bebriefly summarized as follows: A gene encoding a variable domain of amonoclonal antibody of whatever specificity is cloned. The DNA segmentsencoding the framework and hypervariable regions are determined and theDNA segments encoding the hypervariable regions are removed so that theDNA segments encoding the framework regions are fused together withsuitable restriction sites at the junctions. The restriction sites maybe generated at the appropriate positions by mutagenesis of the DNAmolecule by standard procedures. Double stranded synthetic CDR cassettesare prepared by DNA synthesis according to the sequences givenCDR1-11C7, CDR2-11C7, CDR3-11C7, CDR1′-11C7, CDR2′-11C7 and CDR3′-11C7above. These cassettes are provided with sticky ends so that they can beligated at the junctions to the framework by standard protocol forachieving a DNA molecule encoding an immunoglobulin variable domain.

Furthermore, it is not necessary to have access to the mRNA from aproducing hybridoma cell line in order to obtain a DNA construct codingfor the monoclonal antibodies of the invention. Thus POT application WO90/07861 gives full instructions for the production of a monoclonalantibody by recombinant DNA techniques given only written information asto the nucleotide sequence of the gene.

The method comprises the synthesis of a number of oligonucleotides,their amplification by the PCR method, and their splicing to give thedesired DNA sequence.

Expression vectors comprising a suitable promoter or genes encodingheavy and light chain constant parts are publicly available. Thus, oncea DNA molecule of the invention is prepared it may be convenientlytransferred in an appropriate expression vector.

DNA molecules encoding single chain antibodies may also be prepared bystandard methods, for example, as described in WO 88/1649.

In a particular embodiment of the invention, the recombinant means forthe production of some of the Binding Molecules of the inventionincludes first and second DNA constructs as described below:

The first DNA construct encodes a heavy chain or fragment thereof andcomprises

-   a) a first part which encodes a variable domain comprising    alternatively framework and hypervariable regions, said    hypervariable regions comprising in sequence DNA-CDR1-11C7 (SEQ ID    NO: 15), DNA-CDR2-11C7 (SEQ ID NO: 16) and DNA-CDR3-11C7 (SEQ ID NO:    17); this first part starting with a codon encoding the first amino    acid of the variable domain and ending with a codon encoding the    last amino acid of the variable domain, and-   b) a second part encoding a heavy chain constant part or fragment    thereof which starts with a codon encoding the first amino acid of    the constant part of the heavy chain and ends with a codon encoding    the last amino acid of the constant part or fragment thereof,    followed by a non-sense codon.

Preferably, the second part encodes the constant part of a human heavychain, more preferably the constant part of the human γ4 chain. Thissecond part may be a DNA fragment of genomic origin (comprising introns)or a cDNA fragment (without introns).

The second DNA construct encodes a light chain or fragment thereof andcomprises

-   a) a first part which encodes a variable domain comprising    alternatively framework and hypervariable regions; said    hypervariable regions comprising in sequence DNA-CDR1′-11C7 (SEQ ID    NO: 17), DNA-CDR2′-11C7 (SEQ ID NO: 18) and DNA-CDR3′-11C7 (SEQ ID    NO: 19), this first part starting with a codon encoding the first    amino acid of the variable domain and ending with a codon encoding    the last amino acid of the variable domain, and-   b) a second part encoding a light chain constant part or fragment    thereof which starts with a codon encoding the first amino acid of    the constant part of the light chain and ends with a codon encoding    the last amino acid of the constant part or fragment thereof    followed by a non-sense codon.

Preferably, the second part encodes the constant part of a human lightchain, more preferably the constant part of the human κ chain.

The first or second DNA construct advantageously comprises a third partwhich is located upstream of the first part and which encodes part of aleader peptide; this third part starting with the codon encoding thefirst amino acid and ending with the last amino acid of the leaderpeptide. This peptide is required for secretion of the chains by thehost organism in which they are expressed and is subsequently removed bythe host organism. Preferably, the third part of the first DNA constructencodes a leader peptide having an amino acid sequence substantiallyidentical to the amino acid sequence of the heavy chain leader sequenceas shown in SEQ ID NO: 21 (starting with the amino acid at position −19and ending with the amino acid at position −1). Also preferably, thethird part of the second DNA construct encodes a leader peptide havingan amino acid sequence as shown in SEQ ID NO: 23 (light chain, startingwith the amino acid at position −18 and ending with the amino acid atposition −1).

Each of the DNA constructs are placed under the control of suitablecontrol sequences, in particular under the control of a suitablepromoter. Any kind of promoter may be used, provided that it is adaptedto the host organism in which the DNA constructs will be transferred forexpression. However, if expression is to take place in a mammalian cell,it is particularly preferred to use the promoter of an immunoglobulingene.

The desired antibody may be produced in a cell culture or in atransgenic animal. A suitable transgenic animal may be obtainedaccording to standard methods which include micro injecting into eggsthe first and second DNA constructs placed under suitable controlsequences transferring the so prepared eggs into appropriatepseudo-pregnant females and selecting a descendant expressing thedesired antibody.

When the antibody chains have to be produced in a cell culture, the DNAconstructs must first be inserted into either a single expression vectoror into two separate but compatible expression vectors, the latterpossibility being preferred.

Accordingly, the invention also provides an expression vector able toreplicate in a prokaryotic or eukaryotic cell line which comprises atleast one of the DNA constructs above described.

Each expression vector containing a DNA construct is then transferredinto a suitable host organism. When the DNA constructs are separatelyinserted on two expression vectors, they may be transferred separately,i.e. one type of vector per cell, or co-transferred, this latterpossibility being preferred. A suitable host organism may be abacterium, a yeast or a mammalian cell line, this latter beingpreferred. More preferably, the mammalian cell line is of lymphoidorigin e.g. a myeloma, hybridoma or a normal immortalized B-cell, butdoes not express any endogeneous antibody heavy or light chain.

It is also preferred that the host organism contains a large number ofcopies of the vectors per cell. If the host organism is a mammalian cellline, this desirable goal may be reached by amplifying the number ofcopies according to standard methods. Amplification methods usuallyconsist of selecting for increased resistance to a drug, said resistancebeing encoded by the expression vector.

In another aspect of the invention, there is provided a process forproducing a multi-chain binding molecule of the invention, whichcomprises (i) culturing an organism which is transformed with the firstand second DNA constructs of the invention and (ii) recovering an activebinding molecule of the invention from the culture.

Alternatively, the heavy and light chains may be separately recoveredand reconstituted into an active binding molecule after in vitrorefolding. Reconstitution methods are well-known in the ad; Examples ofmethods are in particular provided in EP 120 674 or in EP 125 023.Therefore a process may also comprise

-   (i) culturing a first organism which is transformed with a first DNA    construct of the invention and recovering said heavy chain or    fragment thereof from the culture and-   (ii) culturing a second organism which is transformed with a second    DNA construct of the invention and recovering said light chain or    fragment thereof from the culture and-   (iii) reconstituting in vitro an active binding molecule of the    invention from the heavy chain or fragment thereof obtained in (i)    and the light chain or fragment thereof obtained in (ii).

In a similar manner, there is also provided a process for producing asingle chain or single domain binding molecule of the invention whichcomprises

-   (i) culturing an organism which is transformed with a DNA construct    respectively encoding a single chain or single domain binding    molecule of the invention and-   (ii) recovering said molecule from the culture.

The binding molecules of the invention exhibit very good nerve repairactivity as shown, for example, in the granule cell neurite outgrowthmodel.

1. Granule Cell Neurite Outgrowth Assay (In Vitro)

Neurite outgrowth from dissociated cerebellar granule cells aredetermined as described (Niederöst et al. (1999) J. Neurosci. 19:8979-8989). Briefly, cerebella are removed from decapitated postnatalday 5-7 rats and dissociated by trypsin treatment. To reduce fibroblastcontamination, the cells are preplated onto bacterial dishes. 75,000cells are then cultured per well in 4-well Greiner tissue culture (Huber& Co AG, Rheinach, Basel) dishes (well surface: 1 cm2) in medium(Neurobasal with B27 serum replacement, Invitrogen). Culture dishes arecoated with poly-L-lysine (Sigma). Chaps extracted proteins from totalspinal cord homogenates of adult rats (Spillmann et al. (1998) J. Biol.Chem. 273: 19283-19293) is coated at protein concentrations of 0.5 till8 μg per well over night at 4° C. and washed. The binding molecules ofthe invention are then pre-incubated for 30 min on the test substrateand removed before the cells are added. Cerebellar granule cells areadded and incubated for 24 hours. To stop the experiment, 2 ml of 4%buffered formaldehyde is slowly added to the culture dishes. Culturesare then stained by immunofluorescence for the growth-associated proteinGAP-43 and with Hoechst for cell nuclei (Granule cells are stained withHoechst in order to see if all the cells have neurites (neuritevisualised with anti-GAP-43)). Three pictures are taken randomly at adefined distance of the upper, lower and lateral edge of each well witha 40× object if on a Zeiss Axiophot Fluorescence Microscope. All theneurites in a field are counted on number-coded, randomly arrangedphotographs. The response (outgrowth of the granule cell neurites) isdose-dependent in the range of about 0.1-10 μg total protein per well(the specific activities of a given preparation vary within this range).

Enhancement of neurite outgrowth of cerebellar granule cell in thenon-permissive environment of the above prepared spinal cord extract bypreincubation with a binding molecule of the invention may be observed.E.g. a typical profile for the neutralizing effect of the mouse11C7-IgG1 antibody in the granule cell neurite outgrowth model is givenbelow:

Neurites per field Percentage Assay 1: rat myelin coated at 1 μg perwell no antibody 80.5 100% +mouse IgG 86.5 108% 11C7 250 μg/ml 160 199%Assay 2: rat myelin (prep. 2) coated at 8 ug per well no antibody 20100% +mouse IgG 17.3 86.5%  11C7 250 μg/ml 31 155% 11C7 75 μg/ml 26 130%11C7 7.5 μg/ml 26 130%

The neutralizing activity of the molecules of the invention may also beestimated by measuring the regenerative sprouting and neurite outgrowthin the in vivo spinal cord injury model as follows:

2. Spinal Cord Injury Model (In Vivo)

Adult Lewis rats are injured microsurgically by transecting the dorsalhalf of the spinal cord bilaterally at the level of the 8^(th) thoracicvertebra. Laminectomy, anesthesia and surgery are described in Schnelland Schwab 1993 (Eur. J. Neurosol. 5: 1156-1171). Controls or bindingmolecules of the invention are applied in two different ways: either byimplanting 10⁶ freshly harvested hybridoma cells into one side of thecerebral cortex (grafted animals) or, alternatively, by an implantedintraventricular canula linked to a subcutaneously implanted 2 ml Alzet(Alza Corporation, Palo Alto) pump (pump animals). —Hybridoma graftedanimals: Rats are immunosuppressed for 7-10 days with cyclosporin A andsacrificed by transcardial perfusion with 4% buffered formalin 14 daysafter injury. —Pump animals: Binding molecules of the invention (e.g. at3.3 mg/ml for mouse 11C7) are filled into 2 ml pumps delivering 0.5 μl/hinto the lateral ventricle for 2 weeks. Pumps are implanted at the timeof the spinal cord lesion, and rats are sacrificed 2 weeks later.

Neuroanatomical tracing: The motor and sensory corticospinal tract istraced by injecting the anterograde tracer biotin dextran amine (BDA)into the cortex of the side opposite to the pump or the graft. BDA istransported to the spinal cord within 10-14 days and visualized usingdiaminobenzidine (DAB) as a substrate as described in Brösamle et al.,(2000 J. Neurosci. 20: 8061-8068).

Evaluation of anatomical results: Two methods of evaluation are used: asemi-quantitative and a quantitative one. Semi-quantitative estimationof intensity of sprouting and regeneration: Complete sagittal sectionseries of number-coded, randomly mixed animals are evaluated for thepresence and density of regenerating sprouts rostral to the lesion usingthe following definitions: regenerative sprouts are fibers emanatingfrom the transected CST; they are long, irregular in their course, muchless branched than the normal grey matter collaterals, and they growthtowards and ventrally or laterally around the lesion. Regenerativesprouts often end in a growth cone which can be small and bulbouse orlarge and branched. Density of sprouting is rated on a scale of 0-3 foreach animal. —Long distance regeneration: fibers that can be followedthrough the lesion into the caudal spinal cord are consideredlong-distance regenerating fibers. Their maximal distance from thelesion site can be measured, but is often a minimal distance as someunlesioned fibers from the small ventral funiculus CST are oftenpresent; their branches mix with those of regenerating axons and makedistinction difficult.

Fiber counts (quantitative assay): A line positioned at −0.5 mm rostralto the end of the transected CST is posed on alternating sections of thegrey matter, and all intersections with CST fibers (normal collateralsor sprouts) are counted. Similar lines are positioned caudal to thelesion at a distance of +0.5, +2 and +5 mm from the lesion center.Intersecting fibers are counted and the 3 levels are added to a sumreflecting CST fibers in the caudal spinal cord. These caudal fibers aredivided by the number of fibers −0.5 mm rostral to the CST end to obtaina ratio.

Two weeks after a spinal cord injury destroying about 40% of the spinalcord segment T8, mainly in the dorsal half, including both main CSTs:tracing of the CST in control animals show a moderate degree of reactivesprouting of the tract. This phenomenon corresponds to the spontaneoussprouting in response to injury well known in the literature. Injuredrats being treated with the binding molecules of the invention or withpumps delivering the binding molecules of the invention may show anenhanced sprouting at the lesion site and regeneration of damaged axonsneurite outgrowth of damaged neurites.

Therefore the invention also provides

-   (i) the use of the binding molecules of the invention in the nerve    repair of a mammalian nervous system, in particular human nervous    system,-   (ii) a method of repairing nerves of a mammalian nervous system, in    particular human nervous system which comprises administering an    effective amount of the binding molecules of the invention to a    patient in need of such treatment, or-   (iii) a pharmaceutical composition for nerve repair of a mammalian    nervous system, in particular human nervous system which comprises    the binding molecules of the invention and a pharmaceutically    acceptable carrier or diluent.

In particular, the binding molecules of the invention are useful foraxonal regeneration and improved sprouting after nerve fiber damage.Thus the molecules of the invention have a wide utility in particularfor human subjects. For example the binding molecule of the inventionare useful in the treatment of various diseases of the peripheral (PNS)and central (CNS) nervous system, i.e. more particularly inneurodegenerative diseases such as Alzheimer disease, Parkinson disease,Amyotrophic lateral sclerosis (ALS), Lewy like pathologies or otherdementia in general, diseases following cranial, cerebral or spinaltrauma, stroke or a demyellating disease. Such demyelinating diseasesinclude, but are not limited to, multiple sclerosis, monophasicdemyelination, encephalomyelitis, multifocal leukoencephalopathy,panencephalitis, Marchiafava-Bignami disease, pontine myelmolysis,adrenoleukodystrophy, Pelizaeus-Merzbacher disease, Spongy degeneration,Alexander's disease, Canavan's disease, metachromatic leukodystrophy andKrabbe's disease. In one example, administration of the bindingmolecules of the invention can be used to treat a demyelinating diseaseassociated with NogoA protein. In another example, cells which expressthe binding molecules of the invention may be transplanted to a sitespinal cord injury to facilitate axonal growth throughout the injuredsite. Such transplanted cells would provide a means for restoring spinalcord function following injury or trauma. Such cells could includeolfactory ensheathing cells and stem cells of different lineages offetal nerve or tissue grafts.

In addition, the Binding Molecules of the invention are useful for thetreatment of degenerative ocular disorders which may directly orindirectly involve the degeneration of retinal or corneal cellsincluding ischemic retinopathies in general, anterior ischemic opticneuropathy, all forms of optic neuritis, age-related maculardegeneration, diabetic retinopathy, cystoid macular edema (CME),retinitis pigmentosa, Stargardt's disease, Best's vitelliform retinaldegeneration, Leber's congenital amaurosis and other hereditary retinaldegenerations, pathologic myopia, retinopathy of prematurity, andLeber's hereditary optic neuropathy, the after effects of cornealtransplantation or of refractive corneal surgery, and herpes keratitis.

Furthermore, it was shown that NogoA plays a role in psychiatricconditions, in particular schizophrenia and depression. Hence, thebinding molecules of the invention are useful for the treatment ofpsychiatric conditions, in particular schizophrenia and depression.

The Binding Molecules of the invention can be provided alone, or incombination, or in sequential combination with other agents. Forexample, the binding molecules of the invention can be administered incombination with anti-inflammatory agents such as but not limited tocorticosteroids following stroke or spinal cord injury as a means forblocking further neuronal damage and inhibition of axonal regeneration,Neurotrophic factors such as NGF, BDNF or other drugs forneurodegenerative diseases such as Exelon™ or Levodopa. As used herein,two agents are said to be administered in combination when the twoagents are administered simultaneously or are administered independentlyin a fashion such that the agents will act at the same time.

For the treatment of psychiatric conditions, in particular schizophreniaor depression, the Binding Molecules of the invention can be providedalone or in combination in particular with other agents selected fromthe group consisting of (a) anti-epileptic drugs selected frombarbiturates and derivatives thereof, benzodiazepines, carboxamides,hydantoins, succinimides, valproic acid and other fatty acid derivatesand other anti-epileptic drugs, (b) conventional antipsychotics, (c)atypical antipsychotics and (d) antidepressants.

The term “barbiturates and derivatives thereof” as used herein includes,but is not limited to Phenobarbital and primidon. The term“benzodiazepines” as used herein includes, but is not limited toclonazepam, diazepam and lorazepam: The term “carboxamides” as usedherein includes, but is not limited to carbamazepine, oxcarbazepine and10-hydroxy-10,11-dihydrocarbamazepine. The term “hydantoins” as usedherein includes, but is not limited to phenyloin. The term“succinimides” as used herein includes, but is not limited toethosuximide and mesuximide. The term “valproic acid and other fattyacid derivates” as used herein includes, but is not limited to valproicacid sodium salt, tiagabine hydrochloride monohydrate and vigrabatrine.The term “other anti-epileptic drugs” as used herein includes, but isnot limited to levetiracetam, lamotrigine, gabapentin and felbamate.

The term “conventional antipsychotics” as used herein includes, but isnot limited to haloperidol and fluphenazine.

The term “atypical antipsychotics” as used herein relates to clozaril,risperidone, olanzapine, quetiapine, ziprasidone and aripiprazol.

The term “antidepressants” as used herein includes, but is not limitedto selective serotonin reuptake inhibitors (SSRI's), or selectiveserotonin and norepinephrine reuptake inhibitors (SNRI-s). An SSRI'ssuitable for the present invention can be selected from fluoxetine,fluvoxamine, sertraline, paroxetine, citalopram and escitalopram.

The structure of the active ingredients identified by code nos., genericor trade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications). The corresponding contentthereof is hereby incorporated by reference. Any person skilled in theart is fully enabled to identify the active ingredients and, based onthese references, likewise enabled to manufacture and test thepharmaceutical indications and properties in standard test models, bothin vitro and in vivo.

For the indications mentioned above, the appropriate dosage will, ofcourse, vary depending upon, for example, the particular molecule of theinvention to be employed, the mode of administration and the nature andseverity of the condition being treated. The Binding Molecules of theinvention are conveniently administered by pumps or injected astherapeutics at the lesioned site, e.g. they can be administereddirectly into the CNS intracranially or into the spine intrathecally tothe lesioned site.

Pharmaceutical compositions of the invention may be manufactured inconventional manner. E.g. a composition according to the inventioncomprising the molecules of the invention is preferably provided inlyophilized form. For immediate administration it is dissolved in asuitable aqueous carrier, for example sterile water for injection orsterile buffered physiological saline.

To aid in making up suitable compositions, the binding molecules of theinvention and optionally a second drug enhancing the effect of theBinding Molecules of the invention, may be packaged separately withinthe same container, with instructions for mixing or concomitantadministration. Optional second drug candidates are provided above.

The synergistic effect of a combination of the binding molecules of theinvention and growth factors such as NGF may be demonstrated in vivo bythe spinal cord injury model described above.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Sequence Comparison: Sequence comparison of the NiG fromdifferent species, human, monkey, rat and mouse (SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47 and SEQ ID NO:48, respectively), showing theimmunogenic peptide sequence for the 11C7 mAb.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

In the following examples all temperatures are in degree Celsius (° C.).

The monoclonal antibody of attention in the Examples is a BindingMolecule according to the present invention comprising the variable partof the light chain (SEQ ID NO: 3) and the variable part of the heavychain (SEQ ID NO: 2).

The following abbreviations are used:

ELISA enzyme linked immuno-sorbant assay

FACS fluorescence activated cell sorting.

FITC fluorescein isothiocyanate

FBS foetal bovine serum

HCMV human cytomegalovirus promoter

IgG immunoglobulin isotype

MAb monoclonal antibody

PBS phosphate-buffered saline

PCR polymerase chain reaction

EXAMPLE 1 NiG-D20 (SEQ ID NO: 24) is One of the Neurite OutgrowthInhibitory Fragments of NogoA

Methods:

a) Rat Nogo-A deletion library: Deletion constructs are made usinginternal restriction sites, by ExonuoleaseIII/Mung Bean Nucleasetreatment and by PCR with rat Nogo-A-specific primers on rat Nogo-(method as in WO00/31235): rat Nogo-A (aa 1-1163; DNA as shown hereafterrelated to the amino acids of rat NogoA (SEQ ID NO: 26), e.g. aa 1-1163means that the cDNA construct encodes for polypeptide which starts atthe amino acid1 and ends at amino acid 1163 of the rat polypeptidesequence of NogoA), rat Nogo-B (aa 1-172+976-1163), rat Nogo-C (Nogo-CN-terminal 11 aa+aa 976-1163), rat Nogo-66 (aa 1019-1083), ratGST-Nogo-66 (aa 1026-1091), rat NiR-G (aa 1-979), rat NiR (1-172), ratNiR-D1 (aa 1-31), rat NiR-D2 (aa 59-172), rat NiR-D3 (aa 1-31+59-172),rat EST-Nogo1 (aa 762-1163), rat NiG (aa 174-979), rat NiG-D1(aa174-909), rat NiG-D2 (aa 174-865), rat NiG-D3 (aa 172-723), ratNiG-D4 (aa 172-646), rat NiG-D5 (aa 293-647), rat NiG-D6 (aa 763-975)rat NiG-D7 (aa 174-235+294-979), rat NiG-D8 (aa 218-653), rat NiG-D9 (aa172-259+646-974), rat NiG-D10 (aa 293-979), rat NiG-D11 (aa 209-268),rat NiG-D12 (aa 198-233), rat NiG-D13 (aa 174-216), rat NiG-D14 (aa174-260), rat NiG-D15 (aa 174-190+493-979), rat NiG-D16 (aa174-190+621-979), rat NiG-D17 (aa 174-190+259-979), rat NiG-D18 (aa174-190+263-979), rat NiG-D19 (aa 763-865), rat NiG-D20 (aa 544-725),rat NiG-D21 (aa 812-918), rat NiG-D22 (aa 866-975), rat NiG-D23 (aa914-975), rat NiG-D24 (aa 544-685), rat NiG-D25 (aa 614-725), ratNiG-D26 (aa 544-613), rat NiG-D27 (aa 581-648), rat NiG-D28 (aa614-685), rat NiG-D29 (aa 648-725), rat NiG-D30 (aa 682-725), ratNiG-D31 (aa 544-580), rat NiG-D32 (aa 581-613), rat NiG-D33 (aa614-648), rat NiG-D34 (aa 648-685), rat NiG-D35 (aa 260-556), ratNiG-D36 (aa 260-415). NiR-G and NiR-a are derived from Nogo-A-pET28 byrestriction enzyme digestions. NiG is derived from NiR-G by restrictiondigestion and MungBean Nuclease treatment. NiG-D1, -D3, -D4, -D5, -D7,-D8, -D9, -D10 derived from NiG-pET28 by restriction enzyme digestions.NiG-D15, -D16, -D17, -D18 derived from NiG-pET28 by Exonuclease IIIdigestion. NiR-b, NiR-D1, -D2, -D3 derived by PCR with NIA-a-pET28 as atemplate. NiG-D2, -D6, -D11, -D12, -D13, -D14, -D19, -D20, -D21, -D22,-D23, -D24, -D25, -D26, -D27, -D28, -D29, -D30, -D31, -D32, -D33, -D34,-D35, -D36 derived by PCR using NiG-pET28 as a template. All constructssubcloned into pET28. pET28 used for all the constructs mentioned above.pGEX-6P used for GST-Nogo66 and pET26 for periplasmic expression of ratNiG. Human GST-Nogo-66 (aa 1055-1120 of human Nogo-A) is cloned by PCRon human NogoA DNA (SEQ ID NO: 4) as a template. Deletion constructs arethen cloned into pET28 vector (Novagen), pGEX-6P (Amersham PharmaciaBiotech) and pET26 vector (Novagen). Human GST-Nogo-68 corresponds tothe GST-nogo protein published by GrandPré et al. (supra). Synthetic ratpeptide 4 EELVQKYSNSALGHVNSTIKELRRL (SEQ ID NO: 27) corresponds to thehuman peptide 4 (Human peptide 4 has been shown to be the inhibitoryregion of the Nogo-66 domain (GrandPré et al., 2000)). The orthologousrat peptide has a single mismatch C->S (sea peptide 4 sequence inGrandPré et al., 2000, supra). Synthetic Pro/Ser-rich peptidePSSPPPSSPPPSSPPPS (SEQ ID NO: 28) as well as rat peptide 4 have beenproduced and HPLC-purified by Primm SA. Human NogoA_(—)623-640 (SEQ IDNO: 6) is synthesised and purified by Research Genetics Inc.

b) Generation of human Nogo-A expression constructs. (pRKT-hNogo-A): Ahuman cDNA library constructed in lambda gt10 (Clontech) is screenedwith duplicate filter sets using standard procedures. Fragments of humanNogo-A are amplified by PCR from human whole brain cDNA (Clontech) usinga standard protocol and subsequently cloned into pBluescript, digestedand isolated, or used as screening probes directly. A 400 bp XhoI/SmaIfragment is used as 5′ probe, the 3′ probe is amplified with primersCA-NA-2F: 5′-AAG CAC CAT TGA ATT CTG CAG TTC C-3′ (SEQ ID NO: 29) andCA-NA-3R; 5′-AAC TGC AGT ACT GAG CTC CTC CAT CTG C-3′ (SEQ ID NO: 30).Positive clones are isolated, subcloned and sequence confirmed. Toobtain a full length human Nogo-A cDNA, overlapping clones are assembledusing an unique EcoRI restriction site in the human Nogo-A sequence andsubcloned into Bluescript vector, named Pbsnogoa. To obtainpRK7-hNogo-A, the full length cDNA was inserted into the eukaryoticexpression vector pRK-7 by directional cloning.

c) Generation of human NiG (hNiG) expression plasmids (pET28a-hNiG) forbacterial production: A hNiG encoding DNA fragment is subcloned intoBamHI/XhoI of pET28a (Novagen), after PCR amplification of therespective coding region from Pbsnogoa, in frame with the N-terminalHis- and T7-tag for bacterial expression, using primer sets: forward5′-GTC GCG GAT CCA TGG AGA CCC TTT TTG CTC TTC-3′ (SEQ ID NO: 31);reverse 5′-GTT CTC GAG TTA TGA AGT TTT ACT CAG-3′ (SEQ ID NO: 32). Thefinal plasmid is termed pET28a-hNiG. hNiG was then expressed in E. coliBL21 pRP by induction with 1 mM Isopropyl-beta-D-thiogalactopyranoside(IPGT).

d) Generation of mouse NiG-exon3 (mNiG-exon3) expression plasmid: Theregion encoding mouse exon 3 is amplified from mouse genome BAC templatewith primers: forward 5′-GTG CGG ATC CAT GGA TTT GAA GGA GCA GC-3′ (SEQID NO: 33); reverse 5′-GTT TCT CGA GTG AAG TTT TAT TCA GCT C-3′ (SEQ IDNO: 34) and subcloned into the BamHI/XhoI cloning sites of pET28a. Thefinal plasmid construct is named pET28a-mNiG-exon3.

Cloning of monkey NiG: PolyA RNA is isolated from frozen monkey braintissue and cDNA are synthesised using an oligo dT primer. Twooverlapping fragments covering the 5′ and the 3′ region of the cDNA areamplified by PCR using sequence-specific primers and a proof-readingenzyme. The primers are designed using the known sequence of the humanNiG cDNA. For amplification of the 5′ fragment the primers are5′-TCCACCCCGGCCGCGCCCAA-3′ (SEQ ID NO: 35) and5′-AATGATGGGCAAAGCTGTGCTG-3′ (SEQ ID NO: 36), for the 3′-fragment5′-GGTACAAAGATTGCTTATGAAACA-3′ (SEQ ID NO: 37) andAGCAGGGCCAAGGCAATGTAGG-3′ (SEQ ID NO: 38). The two fragments are thensubcloned and for each fragment at least 4 independent clones weresequenced. The full length cDNA is assembled by overlapping PCR usingthe primers mentioned above and the resulting product is cloned andsequenced again.

e) Production of recombinant NogoNiG proteins and the Nogo-A-deletionlibrary as defined above: The bacterial Nogo-A-deletion library isexpressed in Escherichia coli. Proteins are extracted either by repeatedsonication in sonication buffer (20 mM Tris, 50 mM NaH₂PO₄, 100 mM NaCl,pH 8.0) with 0.75 mg/ml Lysozyme, by solubilisation with B-Per™ (Pierce)or with 8 M urea. NiG expressed with pelB-leader is obtained from theperiplasmic space according to the Novagen protocol for periplasmicprotein purification. Supernatants of pET28-constructs are purifiedusing the Co²⁺-Talon™ Metal Affinity Resin (Clontech) in a batchprocedure. 8 M urea and B-Per™ solubilised lysates are brought tonon-denaturing conditions by increasingly substituting the buffer withsonication buffer during the resin-batch procedure. Proteins are elutedwith 250 mM imidazole in sonication buffer on a gravity column (BioRad).NiG proteins are further purified by gel filtration on Superdex 200HiLoad 16/60. Supernatants of pGEX-6P constructs are purified withG-sepharose column in a batch procedure according to manufacturerindications (Amersham Pharmacia). Cleavage of GST-Nogo-66 is done byincubating solubilised GST-Nogo-66 with PreScission protease andsubsequent HPLC purification. Get electroelution is performed bypreparative SDS-PAGE of IMAC-purified recombinant Nogo and elution withBioRad Electro-Eluter into 50 mM Tris, pH 7.4, 100 mM NaCl, 0.2% (w/v)CHAPS for 1 hr at 250 mA and followed by 30 s of reversed electrodepolarities. Protein concentrations of chromatography-purified proteinsare determined using Pierce Coomassie Stain and BSA as standard protein.Protein concentrations of gel elided proteins are estimated based onband intensity of silver-stained gels (Merril C R, Dunau M L, Goldman D(1981) A rapid sensitive silver stain for polypeptides in polyacrylamidegels. Analyt. Biochem. 110:201-207) with BSA as a standard.

f) Production of recombinant NogoA fragments in CHO cells: A 3119 bpfragment resulting from a partial HincII digest of rat Nogo-A cDNA,NiR-G, is cloned into pSeoTag2 expression vectors (Invitrogen,Groningen, The Netherlands). Transfection of pNiR-G into CHO cellsresults in intracellular, cytoplasmic expression of NiR-G. Stable NiR-GCHO cell lines are selected with 260 μg/ml Zeocin (Invitrogen).Recombinant NiR-G from cell lysate is purified over a Ni²⁺-NTA column(Qiagen AG, Basel, Switzerland). Rat NiG-D20 and Nogo-66 are cloned intopAPtag5 vector by PCR. Transfection of pNiG-D20-AP into CHO cellsresults in NiG-δ20-AP that was secreted into the culture supernatant.Stable pNiG-D20-AP and pNogo-66-AP cell lines were selected with 250μg/ml Zeocin (Invitrogen). Both cell lines are adapted to serum-freemedium (Gibco) conditions and grown in a cell-line chamber (Integra).Supernatants are tenfold concentrated prior to use, and theconcentration of fusion protein is assessed as described elsewhere(Flanagan J G, Leder P (1990) The kit ligand: a cell surface moleculealtered in steel mutant fibroblasts. Cell 63:185-194).

g) 3T3 fibroblast and CHO spreading assays: The 3T3 spreading assays areperformed as described previously (Spillman A A, Bandtlow C E,Lottspeich F, Keller F, Schwab M E (1998) Identification andcharacterization of a bovine neurite growth Inhibitor (bNi-220). J.Biol. Chem. 273:19283-19293). CHO spreading assays are performedessentially the same way as for 3T3 fibroblasts. Briefly, CHO cells aresplit 1:2. 24 hrs later they are trypsinised in PBS-EDTA for 30 s and˜8,000 CHO cells are plated onto culture dishes precoated with 5, 1, 0.5and 0.2 μg/well NiG or Nogo-66. After 30-45 min the cells are fixed with4% (w/v) PFA, 5% (w/v) sucrose and then analysed as described Spillmannet al, supra). ˜100 cells are counted per well with light microscopy;criterion of spreaded cells: (a) attachment to the dish AND (b) extendedmorphology indicative for lamellipodia; under light microscopy the cellsappear darker and larger than not spreaded, round cells; non-spreadedcells are considered those cells that are (a) not attached to the dishOR (b) attached to the dish, but small, rounded, without detectablelamellipodia protruding on the dish. The ratio between spreaded and notspreaded cells defines the degree of non-permissiveness of thesubstratum.

h) PC12 Neurite outgrowth assays: PC12 neurite outgrowth assays areperformed as described previously (Rubin B P, Spillmann A A, Bandtlow CE, Keller F, Schwab M E (1995) Inhibition of PC-12 cell attachment andneurite outgrowth by detergent solubilized CNS myelin proteins. Europ.J. Neurosci. 7: 2524-2629). PC12 cells (a PC12 cell clone able to growindependently of laminin obtained from Moses Chao, New York) are primedfor two days with 50-100 ng/ml NGF (Harlan Bioproducts, Indianapolis) toDMEM, 5% foetal calf serum, 10% horse serum, 100 U/ml Penicillin and 0.5mg/ml Streptomycin (Pen-Strep from Gibco-BRL). PC12 cells are detachedmechanically, trypsinised for 5 minutes with 0.05% trypsin (Sigma) inHBSS (Gibco) and plated at a density of 3,000-5,000 cells/cm2 in culturemedium with 100 ng/ml NGF. Assays were stopped after 24 hrs by adding 4%(w/v) PFA, 5% (w/v) sucrose in PBS, pH8. Cell culture dishes were coatedfor PC12 cells the same way as for 3T3 cells.

i) Retinal ganglion cell stripe assays: The retinal ganglion cell stripeassay is performed according to Vielmetter (see Vielmetter J, Stolze B,Bonhoeffer F, Stuermer C A (1990) in vitro assay to test differentialsubstrate affinities of growing axons and migratory cells. Exp. BrainRes. 81:283-287) with modifications (see Schmalfeldt M, Bandtlow C E,Dours-Zimmermann M T, Winterhalter K H, Zimmermann D R (2000) Brainderived versican V2 is a potent inhibitor of axonal growth. J. Cell Sci.113:807-816). Explants are evaluated after fixation with 4% (w/v) PFA,0.1% (v/v) glutaraldehyde in PBS for 10 min at RT. For immunostainings,fixed explants are blocked for 1 hr at RT with RNO-blocking solution(0.5% (w/v) BSA, 0.3% (w/v) TopBlock (Juro Supply), 0.1% (w/v) NaN₃ inPBS), permeabilized for 10 min with 0.05% (v/v) Tx-100 in RNO-blockingsolution, frozen for one minute at −20° C. and incubated with primaryantibodies (AS Blanca for NiR, AS Laura for Nogo-A, NiR-G, NiG, NiG-D3and NiG-D20, Novagen mAb anti-T7 for Nogo-C and beta-Gal controlprotein). After washing with PBS, FITC- and TRITC (FITC:Fluorescein-IsoThioCyanate: TRITC: Tetramethyl RhodamineIsoThiocyanate)-conjugated antibodies (Jackson ImmunoResearchLaboratories) are added (1:150) to the explants. The samples arecoverslipped in 50% (v/v) glycerol, 25 mM NaHCO₃, 40 mM NaCl, 1% (w/v)p-Phenylenediamine (Sigma).

Results:

a) Two regions in the N-terminal part of Nogo-A are inhibitory forspreading of 3T3 fibroblasts: In order to identify the regions of Nogo-Aresponsible for the inhibition of 3T3 fibroblast spreading, a library of50 Nogo deletion constructs is made and recombinant proteins areexpressed in bacteria (see method 1a). The apparent EC₅₀ for inhibitionof 3T3 fibroblast spreading was approximately 400-500 ng/0.1 ml Nogo-Acoated overnight per cm² of culture dish (˜4 pmol/cm²). Treatment ofNogo-A or its fragments with 8 M urea results in a strong decrease ofinhibitory activity, indicating that conformation is important. Theanalysis of Nogo fragments in the fibroblast spreading assay revealsthat at least two stretches of the Nogo-A protein mediate inhibition ofthe spreading of freshly plated fibroblasts; namely NiR-D2, (aa 59-172)and NiG-D20 (aa 844-725). All the fragments derived from the NiG-regiondisplaying inhibitory activity (e.g. NiG-D4 and NiG-D8) partiallyoverlap with NiG-D20. Minor inhibitory activity at high proteinconcentration is seen for NiG-D19 within the NiG-D6 region. Nogo-C,Nogo-66 and rat Peptide 4 (shown to be the inhibitory region of Nogo-68by GrandPré et al., 2000) are not inhibitory for fibroblast spreading.These data show that the anti-spreading activity of Nogo-A on 3T3fibroblasts resides in two defined stretches located at the N-terminus(NiR-D2) and within the Nogo-A-specific part (NiG-D20) of the protein.Non-specific physico-chemical properties (acidity of the fragments,structural effects due to proline and serine residues) are notresponsible for this effect. The C-terminal RTN domain is not involvedin the inhibition of fibroblast spreading.

b) NiG-D20 Region of Nogo-A is inhibitory for neurite outgrowth: Todetermine whether the fragments of Nogo-A that are non-permissive forcell spreading are also inhibitory for neurite outgrowth, a series ofbacterially produced Nogo-A fragments as well as eukaryotically producedNogo-AP chimeras in different neuronal assays are tested. In the stripeassay (method 1), neurites avoid laminin/Nogo-A coated stripes, growingon the laminin-only stripes, whereas stripes coated withlaminin/beta-Galactosidase are not circumvented. Full-length Nogo-A isstrongly non-permissive for retinal ganglion cell (RGC) neuriteoutgrowth, while the N-terminal part (NiR) had only marginal effects.Nogo-C activity is indistinguishable from the control proteinbeta-Galactosidase. The Nogo-A-specific region NiG-D20 appears tocontain the main region responsible for the non-permissive activity onRGC neurite outgrowth; the growth cones stop when encounteringNiG-D20-coated stripes. The nonpermissive effect isconcentration-dependent. At lower Nogo-A concentrations the number ofcrossing fibers increased. No obvious difference is observed betweennasal and temporal RGC neurites concerning their responsiveness toNogo-A regions. A laminin-independent, NGF-responsive clone of PC12cells is primed with 50 ng/ml NGF for 24 hrs and then plated onto dishescoated with bacterially produced Nogo fragments at 0, 1-3 μg/cm².Neurite outgrowth is scored one day later. The Nogo-A-specific region(NiG) and its fragment NiG-δ20 strongly inhibited PC12 neuriteoutgrowth. In contrast, the N-terminal fragment NiR has only minoractivity, detectable only at high protein concentration. Nogo-C andNogo-66 are inactive.

EXAMPLE 2 Presence of Binding Site(s) for and NiG-D20 on 3T3 Fibroblastsand Rat Cortical Brain Membranes

Methods:

a) Radioactive labelling and binding experiments: IMAC-purified NiG-D20is iodinated by ANAWA Trading SA (Wangen, Switzerland) (2,038 CI/mmol)using Lactoperoxidase and purified by reverse-phase HPLC. Membranes fromrat brain cortex are prepared as described (Olpe H R, Karlsson G, PozzaM F, Brugger F, Steinmann M, Van Riezen H, Fagg G, Hall R G, Froestl W,Bittiger H (1990) CGP 35348: a centrally active blocker of GABABreceptors. Eur. J. Pharmacol. 187:27-38). Binding is performed for 1 hrat RT essentially as described (Kaupmann K, Nugget K, Held J, Flor P J,Bischoff S, Mickel S J, McMaster G, Angst C, Bittiger H, Froestl W,Bettler B (1997) Expression cloning of GABA(B) receptors uncoverssimilarity to metabotropic glutamate receptors. Nature 386:239-246.)using 1.5 ml tubes preincubated for 2 hrs with 1% (w/v) bovine serumalbumin to reduce non-specific binding. Membrane homogenates in HEPESbuffer pH 7.4 (125 mM NaCl, 5 mM KCl, 0.6 mM MgCl₂, 1.8 mM CaCl₂, 20 mMHEPES, 6 mM dextrose) containing protease inhibitors (Rôche Diagnostics,Mannheim, FRG) are incubated with 1.3 nM iodinated NiG-D20 in theabsence or presence of increasing concentrations of unlabelled NiG-D20.

b) How cytometry: Flow cytometry and cell sorting are performed on aCytomation MoFlo high-speed cell sorter (Fort Collins, Colo.). The flowcytometer is equipped with an argon-Ion/UV Enterprise II laser tuned to488 nm with 130 mW of power. Fluorescein (FITC) fluorescence iscollected through a 530/40 nm bandpass filter. For analysis 3T3fibroblasts are detached with Cell Dissociation Buffer (Gibco). Thepre-formed complex used to detect binding of NiR-G to 3T3 fibroblasts isprepared as follows: NiR-G and anti-Myc antibody (9E10) are incubated ata 1:1 molar ratio for 30 min at 4° C. Next, FITC conjugated F(ab)₂ GoatAnti Mouse IgG is added and incubated for additional 30 min at 4° C. Theresulting molar ratio of the trimeric complex is 1:1:0.5. The complex isadded to 1×10⁶ 3T3 fibroblasts in a final volume of 0.1 ml, incubatedfor 2 hrs at 4° C., washed, and analysed by flow cytometry.

Results:

Presence of binding site(s) for Nogo-A-specific active fragments on 3T3fibroblasts and rat cortical brain membranes: Since the NiR-D2 andNiG-D20 regions of Nogo-A are inhibitory for cell spreading and neuriteoutgrowth despite the absence of Nogo-66 and independently of NgR, thepresence of a separate, Nogo-A-specific receptor has to be postulated.Thus binding studies are performed of multimerised, myc-tagged andIMAC-purified NiR-G to living 3T3 fibroblasts that are analysed by flowcytometry. Ab-complexed is binding efficiently to 3T3 cells as seen by afluorescence shift of over 90% of the 3T3 cells. In contrast, 3T3 cellsare not labelled after incubation with the 9E10 primary mouse anti-mycmAb complexed with a FITC-conjugated secondary F(ab)₂ goat anti-mouseIgG nor with the secondary Ab alone. To test binding of NiG-D20 to ratcortical membranes, [¹²⁵I]-labelled NiG-D20 in a radioligand bindingassay is used. At a concentration of 1.3 nM of [¹²⁵I]-NiG-D20, evidencefor a specific NiG-D20 binding sites on brain membranes as shown by aconcentration-dependent competition of radioligand binding by unlabelledNiG-D20 is found. These results show that aminoterminal fragments ofNogo-A can bind to the surface of 3T3 cells and to rat corticalmembranes, demonstrating the presence of membrane-bound, Nogo-A-specificbinding sites or receptor(s).

EXAMPLE 3 Generation of Mouse 11C7-IgG1

Mice (C3H- and C57BI6/J-strains) are immunised subcutaneously with thesynthetic peptide SYDSIKLEPENPPPYEEA (=rat NogoA_(—)623-640; SEQ ID NO:1), corresponding to a particular epitope in NiG-D20. This epitope ishighly conserved in human, cynomologus monkey and mouse NiG-D20 Nogo-Aspecific region and starts at amino acid 623 and ends at amino acid 640of the human NogoA amino acid sequence (SEQ ID NO: 5) (See also sequencealignment: FIG. 1).

mAb 11C7 has been obtained out of a fusion of rat NogoA_(—)623-640 withthe carrier protein Key hole limped hemagglutinin (KLH) immunised mice.Monoclonal antibodies have been screened by ELISA on ratNogoA_(—)623-640-KLH, rat NogoA_(—)623-640 free peptide and a nonrelatedpeptide-KLH. In a further screen, the mAbs have been tested by ELISA onNiR-G versus b-Galactosidase, both expressed as his-tagged proteins andpurified by metal affinity chromatography. Subsequently, the mAbs havebeen tested for recognition of Nogo-A on Western blot of oligodendrocyteand brain lysates (rat origin). Antibodies are tested for recognition ofthe protein in immunocytochemistry of rat Nogo-A-transfected CHO or COScells and of endogenous Nogo-A of rat oligodendrocytes (permeabilizedcells). They have also been tested for surface binding to living ratoligodendrocytes. Species crossreactivity is tested on recombinant NiGof rat, mouse, human and bovine origin by ELISA and on endogenous rat,mouse, human and monkey Nogo-A by Western blot of tissue or cellextracts.

Western blot analysis: SDS-PAGE and Western blotting are performed asdescribed earlier (Huber A B, Weinmann O, Brosamle C, Oertle T, Schwab ME 2002) Patterns of Nogo mRNA and protein expression in the developingand adult rat and after CNS lesions. J. Neurosci. 22: 3553-3567),blocking is done with 3% (w/v) Top Block (Juro Supply, Lucerne,Switzerland). Antibodies are diluted as follows: Purified monoclonal11C7 or hybridoma supernatants 1:150. Secondary antibodies areHAP-conjugate anti-mouse ((Pierce; 1:5000,) 1:50,000). Hybridisationwith the 11C7 antibody is carried out over night at 4° C. For detectionthe ECL detection reagents from Amersham Pharmacia are used.

Results;

The 11C7 mAb identifies the 190 kD Nogo-A band on a Western blot ofoligodendrocyte cell culture homogenate. 11C7 also identifies human NiG,Cynomolgus NiG cell lysate and rat NiG-D20 in western blots. 11C7 mAb ischaracterised as a IgG1 Isotype (IsoStrip Kit, Roche).

EXAMPLE 4 Characterisation of the Mouse 11C7 mAb

Immunocytochemistry: Optic nerve oligodendrocytes are prepared asdescribed (Schwab, Caroni, 1988, Neuron). Three to five day-old culturesgrown on poly-L-lysine coated coverslips are washed twice with PBS,fixed in 4% (w/v) paraformaldehyde (PFA), 5% (w/v) sucrose in PBS for 15min at room temperature (RT) and non-specific binding is blocked with10% (v/v) FCS. Cells were then incubated with mouse 11C7 (1:100).Secondary antibodies are goat-anti-mouse TRITC (Jackson ImmunoResearchLaboratories). For cell surface staining, two day-old rat optic nervecultures are incubated with monoclonal antibody in medium for 25 min atRT. Secondary alkaline phosphatase conjugated antibodies (MilanAnalytica, Lausanne) are used at 1:7,500 in 0.1 M maleic acid with 1%(w/v) blocking reagent (1 hr). The cultures are washed twice with maleicacid buffer, once with alkaline phosphatase buffer (0.1 M Tris-HCl pH9.5, 0.1 M NaCl, 5 mM MgCl₂) and the staining is developed for 3 hrs atroom temperature with 0.175 mg/ml BCIP (Sigma) and 0.338 mg/ml NBT(Sigma) in alkaline phosphatase buffer.

NogoA_(—)623-640 epitope of Nogo-A present at the cell surface ofcultured oligodendrocytes: Living cultures of oligodendrocytes incubatedwith mouse 11C7 mAb stain the differentiated oligodendrocyte cell bodiesand their radial processes. The control mouse IgG and the antibodiesagainst the intracellular protein CNPase do not stain the living cells.Pre-incubation of mouse 11C7 with the corresponding immunogenic peptide(=rat NogoA_(—)623-640 SEQ ID NO: 1) reduces staining to backgroundlevels (competitive assay). Cell surface staining is present on allmajor and small processes and on the cell body. Thus, the Nogo-Aspecific part of the molecule recognised by mouse 11C7 mAb is exposed tothe extracellular space on the plasma membrane of oligodendrocytes.

Production and Purification of mouse 11C7 mAb: A 10-L glass bioreactoris used for continuous-mode cultivation of the hybridoma clone producingthe mouse 11C7 mAb. The bioreactor is equipped with a marine impellerplaced in a center tube for gentle agitation, a spin filter for cellretention, and coiled silicone tubing for bubble-free aeration. Thehybridoma cells are cultivated in our RPMI based serum free medium. Themedium is inoculated with cells at 3.7×10⁵/ml. After 28 hours continuousmedium flow through the bioreactor is started with a rate of 0.6tormentor volumes/day (5 liters/day). Another 24 hours later the flowrate is increased to its final level of 1 fermentor volume/day (10liters/day). After 1 week the culture reaches a steady state with 11×10⁵cells/ml and the process is continued for another week. The titer of themouse 11C7 mAb is determined daily by HPLC. A total of 150 litersculture supernatant is harvested from the bioreactor, sterile filteredfor removal of cells and cell debris. 150 L culture supernatant areconcentrated to about 6 L using a Pellikon tangential flow device(Millipore; 10 kDa cut-off). The concentrated supernatant is purified in3 runs over a 220 ml bed volume column of Protein A Sepharose Cl-4B(Pharmacia; 11 cm bed height). Briefly, the culture supernatant after pHcorrection to 8.1 is loaded at 4 ml/min and the column washed tobase-line at 8 ml/min using 100 mM Na₂HPO₄, pH 8.1. Bound material isfinally eluted at 8 ml/min using 50 mM NaH₂PO₄, pH 3.0, 140 mM NaCl andimmediately neutralized (pH 7.0) with 5 N NaOH and sterile filtered.Absorbance is monitored at 280 nm. Portion of the purified material areeventually further concentrated by ultrafiltration and/or dialyzedagainst PBS. All the buffers used in the purification are filtered on a10 kDa ULTRASETTE™ tangential flow device (Filtron TechnologyCorporation) In order to remove possible endotoxin contaminations. Forthe same reason the Protein A resin is extensively washed with 20%ethanol and all tubings/pumps treated with 0.1 M NaOH prior to use.Protein concentration is measured spectrophotometrically at 280 nm usinga reference absorption of 1.35 for 1 mg/ml. Purity is routinely assessedby SDS-PAGE under reducing conditions using 4-20% Novex gradient gels.Endotoxin content is measured by the classical Limulus Amoebocyte Lysate(LAL) reaction according to the manufacturer instructions (Endotell AG,Allschwil, Switzerland).

Generation of F_(ab) fragments: A portion of mouse 11C7 mAb isextensively dialyzed against 100 mM Na-actetate, pH 5.5, 2 mM EDTA andadjusted to a concentration of 6 mg/ml. F_(ab) fragments are generatedby papain digestion (1:200 w/w ratio) in the presence of 0.25 mMcysteine. The reaction is allowed to proceed for 16 hours at 37° C. andthen stopped by the addition of the specific papain inhibitor E64(N—[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine) in largeexcess (10 μM). The digested antibody is then passed over a column ofprotein A Sepharose Fast Flow in order to remove intact material and Fcfragments. The F_(ab) fraction is extensively dialysed against PBS andconcentrated to about 3 (Papain and E64 are from Roche MolecularBiochemicals).

HPLC, Mass Spectrometry and N-terminal amino acid sequencing of V_(L),and V_(H) regions:

-   a) Reduction and Alkylation: Purified, dried 11C7 antibody are    dissolved in 40 μl of 8M urea, 0.4M NH₄HCO₃, pH 8.3. 60 ug DTT    (Calbiochem), pre-dissolved in 10 ul of the same buffer as the    protein, are added. Reduction is performed at 50° C. for 30 min    under argon (100 fold molar excess of DTT over protein thiols).    After reduction, the sample is cooled to room temperature. 304 ug of    iodoacetamide (Sigma Ultra, I-1149) dissolved in the same buffer as    the protein is added. Carboxamidomethylation is carried out at room    temperature for 15 min in the dark. 1 μl β-mercaptoethanol is added    to quench the reaction.-   b) Isolation of Heavy- and Light-Chain: Carboxamidomethylated heavy    and light chains of antibody are isolated by Reverse Phase High    Pressure Liquid Chromatography (RP-HPLC) on a Hewlett Packard 1090M    HPLC System with DRS pumping system and diode-array UV detector. The    conditions for chromatography are: PerSeptive Biosystems Poros    2.1×100 mm column packed with R1/H material; flow is 0.5 ml/min;    solvents: (A) 0.1% TEA in water and (B) 0.09% TFA/acetonitril/water    9:1; gradient 25-70% B in 8 minutes at 80° C.; detection at 218/280    nm.-   c) LC-ESI-MS: Mass spectrometry is carried out using a Q-Tof    (Micromass, Manchester, UK) quadrupole time-of-flight hybrid tandem    mass spectrometer equipped with a Micromass Z-type electrospray    ionization source (ESI). Acquisition mass range is typically m/z    500-2000. Data are recorded and processed using MassLynx software.    Calibration of the 500-2600 m/z scale is achieved by using the    multiple-charged ion peaks of horse heart myoglobin (MW 16951.5).-   d) HPLC-MS of heavy and light chain: Separation of reduced and    carboxamidomethylated heavy and light chain is performed on a HP1100    HPLC system (Hewlett Packard, Palo-Alto, Calif., USA) employing a 1    mm×150 mm LC Packings column packed with Perseptive Biosystems POROS    R1/H. The column is held at 60° C. Sample volumes of 10 μl are    injected onto the column using a CTC PAL autosampler (CTC, Zwingen,    Switzerland) fitted with a Valco model C6UW HPLC valve (Valco,    Houston, Tex., USA) and a 10 μl injection loop. HPLC was controlled    by MassLynx software (Micromass, Manchester, UK). UV detection is at    214 nm. Eluent A is water containing 0.05% TFA. Eluent B is a 1:9    mixture of water:acetonitrile containing 0.045% TFA. A gradient from    20% B to 90% B is run in 20 minutes at 80° C. The flow rate is    typically 60 μl/min. The total flow from the LC system is introduced    into the UV detection cell, then the ESI source without any    splitting. The HPLC system is controlled and the signal from the UV    detector is processed using MassLynx software (Micromass,    Manchester, UK). The following 5 signals are detected:

TABLE 1 Measured: Signal Interpretation A = 50959.0 Da H-Chain withcarboxamidomethyl-cysteine (CAMCys)* B = 51119.5 Da Signal A + 162 Da(=hexose)** C = 51086.0 Da Signal A + 127 (Lys), H-Chain with CAMCys* D= 51251.0 Da Signal C + 162 Da (=hexose)** E = 24464.8 Da L-Chain withCAMCys *There are two types of H-chain present, one with and one withoutLys at the C-terminal end. The ratio of both forms is approximately50:50%. **Both types of H-chains have two corresponding glycosylatedforms (+162)

-   d) N-terminal amino acid sequencing of V_(L) and V_(H) regions:    Collected H+L chains peaks form HPLC are used for sequence analysis.    Amino acid sequences are determined on a Hewlett Packard G1000A    N-terminal Protein Sequencing System. The system performs automated    Edman chemistry on protein samples retained on miniature adsorptive    biphasic columns. An optimized chemistry method (double couple 3.0)    is used to enhance chemical efficiency, minimize lags and herewith    extend sequence analysis to about 50 residues. Analysis of PTH-amino    acids is performed on an on-line Hewlett Packard HP1090 HPLC System    equipped with a ternary pumping system and a narrowbore (2.1    mm×25 cm) PTH column.    Results:

From mass analysis homogeneous heavy and light chain of mouse 11C7-IgG1are determined. The H-chain is single glycosylated and there are twoforms with a difference on the C-terminal Lysine. Total mass analysis ofheavy and light chain shows a single mass for both chains. HPLCchromatography of mouse 11C7-IgG1 shows a single peak. After HPLCpurification followed by reduction and alkylation pure heavy and lightchain are available. N-terminal sequence degradation is performed onlight-chain and heavy-chain. 45 to 55 amino acids from the N-terminalsequence of L-chain and H-chain are identified by sequence degradation.

Light Chain 1   5   10   15   20   25   30 ▾   ▾   ▾    ▾    ▾    ▾    ▾DVLLTQTPLTLSITIGQPASISCKSSQSLL 31  35  40   45   50   55   60 ▾   ▾   ▾   ▾    ▾    ▾    ▾ HSDGKTYLNWLLQRPGQ Heavy Chain1   5   10   15   20   25   30 ▾   ▾   ▾    ▾    ▾    ▾    ▾EVKLLESGGGLVQPGGSLKLSCVVSGFDFR 31  35  40   45   50   55   60 ▾   ▾   ▾   ▾    ▾    ▾    ▾ RNWMSWVRQAPGKGLEWIGEINPD

EXAMPLE 5 Cloning of the Heavy and Light Chain Genes of Mouse 11C7 mAb

Total RNA is prepared from 10⁷ hybridoma cells (clone 1107) usingTriPure reagent (Roche diagnostics, Germany, Cat.#1667157) according tothe manufacturers instructions. For cDNA synthesis, mRNA is isolatedfrom above prepared total RNA using Oligotex Resin (Qiagen, Germany,cat. #70022).

cDNA is generated by reverse transcription using the followingconditions: 2 μl mRNA, 2 μl 10× reverse transcription buffer, 2 μl(dT)₂₀ primer (10 μM), 0.5 μl RNasin (Promega, 40 U/ml), 2 μl dNTPs (5mM each), 1 μl Omniscript™ reverse transcriptase (Qiagen, Cat #205110),10.5 μl ddH₂O, Reaction: 1 hr at 37° C. For PCR amplification of cDNAencoding for the V_(H) and V_(L) the proofreading enzyme ProofStart™ DNApolymerase is used.

PCR of light and heavy chain: Reaction mix: 2 μl cDNA, 5 μl 10× reactionbuffer, 3 μl dNTPs (5 mM each), 2 μl 5′ primer (10 μM) (see Table 2), 2μl 3′ primer (10 μM) (see Table 2), 1 μl ProofStart (Qiagen, Cat#202203), 36 μl ddH₂O. PCR conditions: 95° C./5 min, (95° C./40 sec, 53°C./1 min, 72° C. 1 min)×35, 72° C./10 min. The resulting PCR productsare ligated directly into pCRbluntTOPO (Invitrogen). The ligation mix istransfected into TOP 10 cells (Invitrogen) and several clones arepicked. The nucleotide sequences of the variable part of the heavy chainof the 11C7 mAb (V-H, SEQ ID NO: 43) and of the light chain of the 11C07mAb (V-L, SEQ ID NO: 44) cDNas are determined on an ABI sequencer. Thesubsequent amino acid sequence of V-H and V-L are shown in SEQ ID NO: 2(V-H) and SEQ ID NO: 3 (V-L). Primers used for PCR amplification of theV_(H) and V_(L) cDNAs; all primers are synthesized by MWG Biotech,Germany.

TABLE 2 SEQ  ID Primer Sequence NO: 5′-V_(L) leaderAATATGAGTCCTGCCCAGTTCCTGTTTC 39 3′-CK TTAGGAATTCCTAACACTCTCCCCTGTTGAAG40 5′-V_(H) leader  AATATGGATTTTGGGCTGATTTTTTTTATTG 41 3′-C_(H) hingeAATTGGGCAACGTTGCAGGTTGACG 42

EXAMPLE 6 Binding of 11C7 and Fab to Nogo-A Domains Using ELISA

Greiner 96 well PS plates (#655161) are coated with 0.4-2 ug/ml Nogoprotein fragments in PBS (100 ul/well) covered and incubated 4 hours atroom temperature. Plates are flicked and refilled with 200 ul/wellblocking buffer (PBS+2% BSA), covered and incubated. 1 h at RT orovernight at 4° C., then washed 4 times with water and PBS. Differentconcentrations of mouse 11C7 mAb or 11C7 Fab are diluted in PBS+2% BSA(100 ul/well), and incubated 2 h at RT or overnight at 4° C. Wash stepis repeated and Goat anti-mouse IgG conjugated with horse radishperoxidase (HRP) at a dilution of 1:5000 (ICN #55550) in PBS/0.1%BSA/0.1% Nonidet 40 (100 ul/well) is added and incubated, 2 h at RT orovernight at 4° C. and wash step is repeated. HRP reaction is started byadding 100 ul/well BM blue POD (Roche #1484281) and incubated in thedark at RT for 15 minutes. H2SO4 50 ul/well 1M is added to stop HRPsubstrate reaction and the optical density is determinated using amicroplate reader (Packard Spectra Count) set to 450 nm.

The mouse 11C7 mAb binds to human NiG, rat NiG, mouse NiG, rat NiG-D20and peptide 472 at very low concentrations of 0.02 to 2.5 nM. Binding tohuman NiG, rat NiG, mouse NiG at very low concentration is confirmed bythe very high affinity (Kd 0.1-0.44 nM Biosensor affinity measurements)and is consistent with the fact that 472 peptide with the exception of2-3 amino acids is identical in human compared to rat and mouseequivalent region. The specificity of the binding is indicated by thefact that the mouse 11C7 mAb does not show any binding at all to ratNiG-D6 and Nogo-66 fragments over the same concentration range. The Fabmonovalent fragment bound to human NiG and rat NiG-D20 at concentrations0.025 to 25 nM and showed no binding to rat NiG-D6 and Nogo-66 fragmentsover the same concentration range. The Kd measured by Biosensor was 7.14nM for human NiG.

EXAMPLE 7 Biosensor Affinity Measurements for Mouse 11C7-IgG1 and Fab toNogo-A Domains

The affinity of the mouse 11C7 mAb and of the 11C7 Fab are measured bysurface plasmon resonance (SPR) using a BIAcore 2000 optical biosensor(Biacore, Uppsala, Sweden) according to the manufacture's instructions(see FIG. 2). Recombinant human, mouse, and rat NiG are covalentlyattached to three separate flow cells of a CMS sensor chip usingamine-coupling chemistry. Briefly; the carboxymethyladed dextran matrixis activated by injecting 35 ul of a solution containing 0.025M NHS and0.1M EDC. For the immobilization on the sensor chip the recombinantmouse, human, and rat NiG are diluted in 0.01 M citrate buffer at a pHvarying between 3.5 and 4.5 and injected at a flow rate of 5 ul/min toachieve coupling levels allowing affinity measurements. The deactivationof the remaining NHS-ester group is performed by injection of 35 ul of1M ethanolamine hydrochloride (pH 8.5): The surface of the sensor chipis regenerated by injecting 5 ul 0.1M HCl. For the measurement of theaffinity the antibodies are injected at different concentration, rangingfrom 0.50 nM to 100 nM at a flow rate of 200 ul/min. After eachinjection the sensor chip surface is regenerated with the injection of10 ul 0.1M HCl without loss of binding activity on the surface. Thekinetic constants, ka and kd and the affinity constants KA and KD areevaluated using the BIAevaluations 3.0 software supplied by themanufacturer.

Affinity measurement in BIAcore: The kinetic and the affinity bindingconstants of the mouse 11C7 mAb and the 11C7 derived monovalent Fabfragment to recombinat NogoA are measured in real time using surfaceplasmon resonance (SPR) technology (Biacore). For this analysisrecombinant human, mouse and rat NiGs are coupled on three independentsensor chip surfaces and different concentrations of the antibodies areinjected. Kinetic parameters of the binding interactions are derivedfrom the sensorgrams by non-linear curve fitting. The affinity constantsat equilibrium of mouse 11C7-IgG1 are KD=0.1 nM, KD=0.4 nM and KD=0.19nM for human, rat, and mouse NiG respectively (table 3). For the 11C7derived Fab fragment the affinity constant to human NiG is KD=7.14 nM.The lower affinity of the Fab fragment results from a decrease of bothkinetic constants, association and dissociation (ka, kd). Lower affinityof the Fab fragment compared to the complete antibody is probablyrelated to the avidity effect, which is lacking in the monomeric Fab.

TABLE 3 Ka (1/Ms) kd (1/s) KA (M⁻¹) KD (M) 11C7 HumanNIG 4.48 × 10⁵  4.6× 10⁻⁵ 9.73 × 10⁹ 1.03 × 10⁻¹⁰ Rat NIG 8.76 × 10⁵ 3.89 × 10⁻⁴ 2.25 × 10⁹4.44 × 10⁻¹⁰ Mouse NIG 5.52 × 10⁵ 1.06 × 10⁻⁴  5.2 × 10⁹ 1.92 × 10⁻¹⁰11C7 Fab HumanNIG 7.29 × 10⁴ 5.28 × 10⁻⁴  1.4 × 10⁸ 7.14 × 10⁻⁹

The invention claimed is:
 1. A method for inducing or enhancing nerverepair comprising administering to a subject in need of such treatmentan isolated human Nogo A623-640 binding molecule which comprises a heavychain variable domain or fragment thereof, and a light chain variabledomain or fragment thereof, wherein said heavy chain variable domain orfragment thereof, comprises in sequential order the amino acid sequencesof SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; and wherein said lightchain variable domain or fragment thereof comprises in sequential orderthe amino acid sequences of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.2. A method of treatment of spinal chord injury in a subject in need ofsuch treatment comprising administering to the subject an isolated humanNogo A623-640 binding molecule which comprises a heavy chain variabledomain or fragment thereof, and a light chain variable domain orfragment thereof, wherein said heavy chain variable domain or fragmentthereof, comprises in sequential order the amino acid sequences of SEQID NO:8, SEQ ID NO:9 and SEQ ID NO:10; and wherein said light chainvariable domain or fragment thereof comprises in sequential order theamino acid sequences of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.